We propose to set up cell systems transformed for restricted regions of the HSV-1 genome which will be analyzed biochemically and genetically for information on viral gene expression and regulation. Transformation will be attempted by cotransfection of tk- cells using tk+ selection and by transformation without selection by delivery of HSV-1/pBR325 chimeric plasmids directedly to Vero cells as a consequence of fusion with bacterial protoplasts. The protoplast fusion method will be tested for more efficient induction of morphological transformation. To help define the HSV-1 genome, mutants will be induced, isolated, mapped and characterized. Temperature-sensitive mutations will be induced in HSV-1 DNA by mutagenic treatment of bacterial cells carrying chimeric plasmids for specific restriction enzyme fragments. Cells transformed for HSV-1 restriction fragments will be used as host range systems for the isolation of deletion mutants of the virus. Two new methods of marker rescue will be tested. These are a protoplast fusion-infection technique and superinfection with cells transformed with specific restriction enzyme fragments. Mutational saturation of two HSV-1 restriction enzyme fragments EcoRI-Fanol-H will be attempted. Correlations between physical and genetic maps will be made. In an attempt to gain insight into HSV-1 latency, the interaction of HSV-1 KOS with a neuronal cell, which results in high frequency cell survival with persistence of viral genetic sequences will continue to be studied. Hybridization experiments will be carried out which should distinguish between the persistence of the entire viral genome or only a portion of it in the neuronal survivors.